Chromatin immunoprecipitation and gene expression analysis of neuronal subtypes after fluorescence activated cell sorting

Abstract

Background With advances in cell capture, gene expression can now be studied in neuronal subtypes and single cells; however, studying epigenetic mechanisms that underlie these changes presents challenges. Moreover, chromatin immunoprecipitation (ChIP) protocols optimized for low cell number do not adequately address technical issues and cell loss while preparing tissue for fluorescence activated cell sorting (FACS). Developing a reliable FACS-ChIP protocol without the need for pooling tissue from multiple animals would enable study of epigenetic mechanisms in neuronal subtypes. Methods FACS was used to isolate dopamine 1 receptor (D1R) expressing cells from the nucleus accumbens (NAc) of a commercially available BAC transgenic mouse strain. D1R+ cells were used to study gene expression as well as histone modifications at gene promoters using a novel native ChIP protocol. Results Isolated cells had enrichment of the dopamine 1 receptor (D1R) mRNA and nearly undetectable levels of GFAP and D2R mRNA. ChIP analysis demonstrated the association of activating or repressive histone modifications with highly expressed or silent gene promoters, respectively. Comparison with existing methods: The ChIP protocol developed in this paper enables characterization of histone modifications from ~30,000 FAC-sorted neurons. Conclusions We describe a one day FACS-ChIP protocol that can be applied to epigenetic studies of neuronal subtypes without pooling tissue.

Cell-Type-Specific Epigenetic Editing at the Fosb Gene Controls Susceptibility to Social Defeat Stress

“…A cell-type-specific examination of Fosb-specific histone modifications would greatly enhance our understanding of the relevance of global vs gene-specific histone modifications within specific cell populations. Technical innovations that enable ChIP following fluorescence-activated cell sorting (FACS) (Finegersh and Homanics, 2016;Mitchell et al, 2017;von Schimmelmann et al, 2016) as well as single-cell ChIP sequencing (Rotem et al, 2015) are necessary to elucidate the cell-type-specific mechanisms of Fosb gene expression in regulating stress-and depressive-like behavior.…”

Single sample sequencing (S3EQ) of epigenome and transcriptome in nucleus accumbens

“…Because the S3EQ method involves extraction of both chromatin and RNA from the same tissue, nuclear RNA cannot be recovered. This is an especially prominent issue given the high number of transcriptomic studies of FACS sorted neuronal nuclei (Guo et al 2014; Cai et al 2014; Finegersh & Flomanics 2015; Lacar et al 2016; Jiang et al 2015). To examine the potential relevance of nuclear or cytosolic RNA loss, we carried out RNA-seq on RNA isolated from either cytosol alone, nuclear-enriched fraction alone, or whole cell.…”

“…While FACS addresses the issue of cellular heterogeneity, it is unclear if RNA-seq of nuclei alone can capture the full extent of gene expression in bulk tissue samples that include nuclei, soma and processes. A second major limitation of FACS is the relatively low recovery of nuclei, which therefore requires pooling of a large number of animal or human samples (Finegersh & Homanics 2016; von Schimmelmann et al 2016; Jiang et al 2015). Acquisition of large sample cohorts is not always feasible especifally with human tissue, complex animal behavioral studies or low throughput animal disease models, including those cited above (Nott et al 2016; Labonté et al 2017).…”

DNA fragment‐assisted microfluidic chip for capture and release of circulating tumor cells

“…And also, microfluidic chip are gradually emerging as a powerful tool for CTCs enrichment and detection, due to their large surface area‐to‐volume ratio, small sample consumption, and easy integration with other technologies . Excellent performance of these microfluidic devices has been achieved based on a variety of methods, including immunoassay , micro‐filters for size‐based separation , highly dense arrays conjugated with aptamers , magnetic‐activated cell sorter , fluorescence‐activated cell sorter, and so on. Zheng et al.…”